Method for producing dihydroxyacetone-3-phosphate

ABSTRACT

Methods are provided to produce highly concentrated dihydroxyacetone-3-phosphate efficiently by a simple catalytic reaction of dihydroxyacetone with the bacterial cells transformed with the gene encoding a dihydroxyacetone kinase or the dihydroxyacetone kinase produced by said bacterium.

TECHNICAL FIELD

[0001] The present invention relates to a method for producing dihydroxyacetone-3-phosphate using enzyme.

BACKGROUND ART

[0002] Dihydroxyacetone-3-phosphate is a substrate for fructose 1,6-bisphosphate aldolase and is used for stereoselectively synthesizing many kinds of carbohydrates by aldol condensation with various aldehydes. The carbohydrates thus synthesized can be widely utilized as medicines and their synthetic intermediates. The above reaction, however, requires large excesses of expensive dihydroxyacetone-3-phosphate, making the products expensive.

[0003] For example, dihydroxyacetone-3-phosphate can be chemically produced by directly phosphorylating dihydroxyacetone dimer with phosphorus oxychloride (POCl₃) in pyridine (Tetrahedron Lett., 28, 1641 (1987)). This production method is complicated and thus cannot produce dihydroxyacetone-3-phosphate in a high yield at low cost.

[0004] Methods of producing dihydroxyacetone-3-phosphate using enzymes have also been reported. An example thereof is a method of synthesizing dihydroxyacetone-3-phosphate from dihydroxyacetone with immobilized glycerol kinase (GK) under conditions that ATP is regenerated by using phosphoenolpyruvic acid and pyruvate kinase (PK) (J. Org. Chem., 48, 3199(1983), and J. Am. Chem. Soc., 107, 7019(1985)). In this method, acetate kinase and acetylphosphate may be used instead of phosphoenolpyruvic acid and pyruvate kinase (J. Am. Chem. Soc., 107, 7019(1985)). These methods using glycerol kinase (GK) require highly purified enzymes and therefore cannot produce dihydroxyacetone-3-phosphate in a high yield at low cost.

[0005] For another example, it is theoretically possible to produce dihydroxyacetone-3-phosphate from dihydroxyacetone using dihydroxyacetone kinase (DHAK) (examined published Japanese patent applications (JP-B) No. Hei 4-29349 and Hei 4-22560). In particular, dihydroxyacetone kinase derived from yeast Schizosaccharomyces pombe is suitable for producing dihydroxyacetone-3-phosphate (JP-B Hei 4-29349). However, the cell extract of Schizosaccharomyces pombe contains coexisting enzymes such as phosphatases and triose-3-phosphate isomerase, which degrade the formed dihydroxyacetone-3-phosphate. The enzyme thus needs to be highly purified and has not yet been applied practically.

[0006] For the practical production of dihydroxyacetone-3-phosphate using enzymes, the enzyme or the biocatalyst containing the enzyme should have high reactivity, be prepared easily at low cost, and be highly purified so as not to degrade the product, dihydroxyacetone-3-phosphate. No efficient method for producing dihydroxyacetone-3-phosphate by using enzymes or biocatalysts containing the enzymes, which meets the above requirements, has been developed.

DISCLOSURE OF THE INVENTION

[0007] An objective of this invention is to provide a method for efficiently producing dihydroxyacetone-3-phosphate.

[0008] Under the above circumstances, the present inventors have attempted to clone a gene encoding dihydroxyacetone kinase and to express it efficiently to obtain the practical enzyme. The present inventors have succeeded in overexpressing the enzyme in a microbial host. Furthermore, the present inventors have found that dihydroxyacetone-3-phosphate can be produced in a high concentration by contacting the host cells as they are or the enzyme produced by them with dihydroxyacetone, the substrate of the enzyme, thereby accomplishing the present invention.

[0009] More specifically, this present invention relates to:

[0010] (1) a method for producing dihydroxyacetone-3-phosphate, which comprises contacting dihydroxyacetone with bacterial cells transformed with the gene encoding dihydroxyacetone kinase or the enzyme produced by the bacterial cells;

[0011] (2) a method described in (1), wherein said dihydroxyacetone kinase is derived from yeast belonging to the genus Schizosaccharomyces;

[0012] (3) a method described in (1), wherein said dihydroxyacetone kinase is derived from Schizosaccharomyces pombe;

[0013] (4) a method described in (1), wherein said bacterial cell is Escherichia coli; and

[0014] (5) a method described in any one of (1) to (4), wherein said contacting is performed under the conditions that coenzyme, ATP, is regenerated.

[0015] The source of the gene encoding dihydroxyacetone kinase used in the present invention is not limited. It is preferably derived from yeast belonging to the genus Schizosaccharomyces; more preferably, Schizosaccharomyces pombe; and still more preferably, Schizosaccharomyces pombe IFO 0354. The nucleotide sequence of the dihydroxyacetone kinase gene from Schizosaccharomyces pombe IFO 0354 is shown in SEQ ID NO: 1, and the deduced amino acid sequence is shown in SEQ ID NO: 2.

[0016] In the method of this invention, microorganisms transformed with dihydroxyacetone kinase capable of overexpressing the enzyme can be used as they are as an enzyme catalyst. The term “overexpression” as used herein means a higher level expression than spontaneous expression. In general, transformants capable of overexpressing the expression product can be obtained by introducing multiple copies of the expression unit with the gene encoding the expression product into host cells. The host bacterial cells used in the present invention are not particularly limited. It is preferable to use bacteria belonging to the genus Escherichia, and more preferable to use E. coli K-12 strain because various host-vector systems can be used. For example, pUC118 or pUC119 is the preferable expression vector into which the dihydroxyacetone kinase gene is to be inserted (Methods in Enzymology, 153, 3 (1987)). The gene can be introduced into the host cell by methods well known in the art such as a method described in “Methods in Enzymology, 68, 299 (1979).” For example, E. coli capable of overexpressing dihydroxyacetone kinase can be prepared as described below. First, the primers are designed for polymerase chain reaction (PCR) based on the database resulting from genome analysis of Schizosaccharomyces pombe. The target DNA is amplified by PCR using the primers and genomic DNA isolated from Schizosaccharomyces pombe IFO 0354 as a template DNA by conventional procedures. The PCR products are ligated into appropriate vectors and are cloned. The gene-inserted vectors are transformed into an appropriate strain of E. coli, and the transformants are then selected by detecting the existence of drug resistant gene(s) and the DNA insert(s). The DHAK productivity of the clones is measured by assaying the activity of the enzyme.

[0017] The enzyme produced by the bacterial cells can be isolated from the cells for use in the method of the present invention. The enzyme is not necessarily purified. A crude enzyme is used as well as the purified enzyme. The dihydroxyacetone kinase can be recovered from the grown cells by extracting the enzyme from the cells first because the enzyme is produced intracellularly. Namely, the cells are collected from the culture medium by filtration or centrifugation and disrupted by mechanical methods such as treatment with alumina, dynomill, or ultrasonication to extract the enzyme in a soluble form. Alternatively, the cell membranes are destroyed by treatment with organic solvents such as acetone, and the resulting cells are dried under reduced pressure. The powder thus prepared is used as a catalyst containing the enzyme. Insoluble materials are removed from the culture obtained in the methods described above by filtration or centrifugation to obtain the crude enzyme. Furthermore, the crude enzyme is concentrated and purified by methods known in the art such as adsorption chromatography, ion exchange chromatography, and gel filtration chromatography. The thus-obtained, partially purified preparation of dihydroxyacetone kinase is also used in the method of this invention.

[0018] The dihydroxyacetone kinase activity of the E. coli strain that overexpresses the enzyme or the enzyme isolated from said cells is measured by known methods (JP-B Hei 04-29349) as follows. The decrease of absorbance at 340 nm is spectrophotometrically measured during the reaction at 25° C. in 1 ml of the reaction mixture containing 0.1M triethanolamine buffer (pH 7.5), 2.5 mM ATP, 4 mM MgSO₄, 0.2 mM NADH, 2.5 units of glycerol-3-phosphate dehydrogenase (G3PDH), and 0.01 ml of a test enzyme solution (formula below).

[0019] One unit of the enzyme of the present invention is defined as the amount required to decrease 1 μmole of NADH in 1 min under the conditions described above.

[0020] Dihydroxyacetone-3-phosphate is usually produced by the method of this invention under the following conditions. First, the reaction is carried out in a buffer with pH ranging from 7 to 8. Any buffer solution can be used as long as it can keep its pH within the above reaction pH range. Tris-hydrochloride buffer is preferable. The reaction temperature may be any range as long as dihydroxyacetone kinase used is active. Preferably, the temperature ranges from about 20° C. to about 35° C. It is necessary to add ATP to the reaction mixture since dihydroxyacetone kinase requires ATP as a coenzyme. However, the enzyme is inhibited by the formed ADP. Therefore, the reaction is preferably performed with regenerating ATP. Any known systems for regenerating ATP (J. Org. Chem. 48, 3199 (1983) and J. Am. Chem. Soc. 111, 627 (1989)) can be used. In view of the production cost, a system using acetyl phosphate and acetate kinase is preferred.

[0021] Dihydroxyacetone-3 -phosphate produced by the above reaction can be identified by enzymatic and chemical techniques. Namely, the decrease of absorbance at 340 nm is spectrophotometrically measured during the reaction (formula below) at 30° C. in 3 ml of a reaction mixture containing 0.1 M sodium acetate buffer(pH 6.0), 0.25 mM NADH, 0.6 units of glycerol-3-phosphate dehydrogenase (G3PDH), and 0.01 ml of a test solution. The concentration of dihydroxyacetone-3-phosphate (DHA phosphate) in the test solution is then determined based on a calibration curve which is made in advance using the standard compound.

[0022] After the above reaction, the reaction product can be precipitated by adding organic solvents such as ethanol to the reaction mixture, or by salting out, to recover it. Furthermore, the reaction product is purified by usual purification procedures such as column chromatography and recrystallization. Alternatively, it can be subjected to the next procedure as it is.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 illustrates the structure of the pUDK vector.

BEST MODE FOR IMPLEMENTING THE INVENTION

[0024] The following examples illustrate the present invention in more detail, but are not to be construed to limit the scope of the present invention.

EXAMPLE 1 Preparing Escherichia coli Strain Overexpressing Dihydroxyacetone Kinase

[0025] The DHAK gene derived from Schizosaccharomyces pombe IFO 0354 was obtained and analyzed in the following manner. Two primers, primer A (5′-GCCGGTACCAGGAGCTAAAATATGGATAAGCAC-3′, SEQ ID NO: 3) and primer B (5′-GCCAAGCTTAAAATTTCGTATCCAATTATTCG-3′, SEQ ID NO: 4) were designed based on the database resulting from the genome analysis of Schizosaccharomyces pombe. Primer A contains the restriction endonuclease KpnI recognition site and E. coli SD sequence, while primer B contains the restriction endonuclease HindIII recognition site. The target gene (dhakI) was amplified by PCR using genomic DNA isolated from Schizosaccharomyces pombe IFO 0354 by a conventional method as a template DNA and the above primers. The PCR products were ligated with pT7BlueT vectors and cloned. The coding region of the desired gene was then isolated from the vector, and ligated with pUC118 vector. The vector thus obtained was named pUDK.

[0026]E. coli strain JM109was transformed with pUDK. The transformant thus obtained was named “E. coli JM109 (pUDK).” Dihydroxyacetone kinase was produced by this transformant as follows. E. coli JM109 (pUDK) was inoculated into LB broth (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0) containing 100 mg/ml of ampicillin, 0.4 mM IPTG (isopropylthiogalactopyranoside) as an inducer and cultured at 37° C. for 16 hours with shaking. FIG. 1 shows the structure of the pUDK vector. SEQ ID NO: 1 shows the nucleotide sequence of dhakI, and SEQ ID NO: 2 shows the deduced amino acid sequence. The molecular weight of this enzyme was estimated to be 145,000 by an HPLC analysis, and the molecular weight of the subunit was estimated to be 63,000 by SDS-polyacrylamide gel electrophoresis. The molecular weight of the subunit agreed well with the molecular weight (62,245) of the deduced amino acid sequence. The requirement of divalent metal cations on the enzyme activity was investigated. The relative activity of the enzyme in the presence of Ca²⁺, Co²⁺, and Mn²⁺ is 116%, 20%, and 12%, respectively, taking the relative activity of the enzyme in the presence of Mg²⁺ as 100%. These results indicate that this enzyme is dimeric and that the cloned dhakI gene encodes the isozyme of dihydroxyacetone kinase I derived from Schizosaccharomyces pombe (JP-B Hei 04-29349).

EXAMPLE 2 Comparison of the Activities of Crude Enzyme Solutions Derived from E. coli JM109 (pUDK) and S. Pombe

[0027]E. coli JM109 (pUDK) was inoculated into LB broth (1% trypton, 0.5% yeast extract, 1% NaCl, pH 7.0) containing 0.4 mM IPTG (isopropylthiogalactoside) and cultured at 37° C. for 16 hours with shaking. The cells were collected from the culture medium by centrifugation and resuspended in a buffer solution. The suspended cells were disrupted by sonication, and the lysate was then centrifuged to obtain the supernatant as a crude enzyme solution. Schizosaccharomyces pombe IFO 0354 was cultured according to the method described in JP-B Hei 04-29349, and the collected cells were disrupted by aluminum oxide to obtain the crude enzyme solution in the same manner as above. The activities (unit/dl of broth) of dihydroxyacetone kinases and contaminating enzymes from both strains are shown in Table 1. TABLE 1 Schizosaccharomyces E. coli JM109 (pUDK) pombe IFO 0354 DHAK 80 14 alkaline phosphatase 0 2.2 triose-3-phoshate 58 485 isomerase

[0028] As clearly shown in Table 1, the crude enzyme solution from E. coli JM109 (pUDK) has high dihydroxyacetone kinase activity and low activities of contaminating enzymes, while the crude enzyme solution from S. pombe IFO 0354 has low dihydroxy kinase activity and high activities of contaminating enzymes. The productivity of dihydroxyacetone-3-phosphate using the crude enzyme solution from both strains was measured by allowing 10 ml of a reaction mixture containing 100 mM DHA, 10 mM MgSO₄, 4 mM ATP, 100 mM acetyl phosphate (AcOP), 6 units of AK (Unitika Co., Ltd), 6 units of DHAK, and 0.1M Tris-HCl buffer (pH 7.3) to react at 25° C. for 10 to 15 hours. As a result, a significant difference was observed in the two. The results are shown in Table 2. TABLE 2 DHAK concentration (mM) Time (hours) E. coli JM1O9 (pUDK) S. pombe IFO 0354 0  0 0 1 32 7 2 41 4 4 63 4 6 70 4 8 63 3 24  55 4

EXAMPLE 3 Producing Dihydroxyacetone-3-phosphate Using a Crude Enzyme Solution from E. coli JM109 (pUDK)

[0029] Ten milliliters of a reaction mixture containing 50 mM DHA, 10 mM MgSO₄, 60 mM ATP, 6 units of dihydroxyacetone kinase, and 0.1 M Tris-HCl buffer (pH 7.3) was allowed to react at 25° C. for 10 to 20 hours to produce 20 mM DHA phosphate (3.8 mg/ml of monosodium dihydroxyacetone-3-phosphate).

EXAMPLE 4 Producing Dihydroxyacetone-3-phosphate Using a Crude Enzyme Solution from E. coli JM109(pUDK) with an ATP Regeneration System

[0030] Ten milliliters of a reaction mixture containing 100 mM DHA, 10 mM MgSO₄, 4 mM ATP, 100 mM acetyl phosphate (AcOP), 6 units of AK (Unitika Co., Ltd), 6 units of a crude DHAK solution, and 0.1 M Tris-HCl buffer (pH 7.3) was allowed to react at 25° C. for 10 to 15 hours, producing 70 mM DHA phosphate (13.3 mg/ml of monosodium dihydroxyacetone-3-phosphate).

EXAMPLE 5 Producing Dihydroxyacetone-3-phosphate Using E. coli JM109 (pUDK) with an ATP Regeneration System

[0031] The reaction was performed in the same manner as in Example 2 except for using E. coli JM109 (pUDK) cells containing 6 units of dihydroxyacetone kinase and produced 73 mM DHA phosphate (13.9 mg/ml of monosodium dihydroxyacetone-3-phosphate).

EXAMPLE 6

[0032] Ten milliliters of a reaction mixture containing 200 mM DHA, 10 mM MgSO₄, 4 mM ATP, 250 mM acetyl phosphate (AcOP), 24 units of AK (Unitika Co., Ltd), 24 units of a crude DHAK solution, and 0.1 M Tris-HCl buffer (pH 7.3) was allowed to react at 25° C. for 15 to 20 hours while controlling the pH; 140 mM DHA phosphate (26.6 mg/ml as monosodium dihydroxyacetone-3-phosphate) was then produced. The reaction is shown in Table 3. TABLE 3 Time (hours) DHAP concentration (mM) 0 0 1 35 2 57 4 81 6 102 8 116 10 133 12 140 24 116

[0033] The reaction mixture was treated with active carbon and filtrated. Barium acetate (0.08 g) was then added to the filtrate, and the resulting precipitate was removed by filtration. The filtrate was concentrated to 3 ml under reduced pressure, then a five-fold volume of cold ethanol was added to the filtrate. The resulting precipitate was filtered and dried under reduced pressure to obtain 180 mg of monosodium dihydroxyacetone-3-phosphate.

Industrial Applicability

[0034] The present invention provides a method for efficiently producing dihydroxyacetone-3-phosphate in a high concentration by using the reaction of dihydroxyacetone kinase.

1 4 1 1743 DNA Schizosaccharomyces pombe CDS (1)..(1743) IFO 0354 1 atg gat aag cac ttt atc aac gat cct gaa gtc ctc gtc ctt gat ggc 48 Met Asp Lys His Phe Ile Asn Asp Pro Glu Val Leu Val Leu Asp Gly 1 5 10 15 ctt aaa tcc ttg gcc gac atg aac aaa act tta act gtt cat gaa gag 96 Leu Lys Ser Leu Ala Asp Met Asn Lys Thr Leu Thr Val His Glu Glu 20 25 30 gga aaa ttc atc tat ttc cat gac tac aac aaa aag aat gtc agt gtc 144 Gly Lys Phe Ile Tyr Phe His Asp Tyr Asn Lys Lys Asn Val Ser Val 35 40 45 att tcc ggc ggt ggt gct ggt cat gaa ccc act cat tct tcg ttc gtg 192 Ile Ser Gly Gly Gly Ala Gly His Glu Pro Thr His Ser Ser Phe Val 50 55 60 ggc aag ggt atg ctt act gcc gcc gtc tca ggc tcc att ttt gct tct 240 Gly Lys Gly Met Leu Thr Ala Ala Val Ser Gly Ser Ile Phe Ala Ser 65 70 75 80 ccg tcg tca aag caa att tat acc ggt att aag caa gtc gaa tct gag 288 Pro Ser Ser Lys Gln Ile Tyr Thr Gly Ile Lys Gln Val Glu Ser Glu 85 90 95 gct ggc acc ttg gta att tgc aaa aac tac acc ggt gac atc ctt cac 336 Ala Gly Thr Leu Val Ile Cys Lys Asn Tyr Thr Gly Asp Ile Leu His 100 105 110 ttt ggt atg gcc ttg gag aag caa aga acg gct ggt aag aag gct gaa 384 Phe Gly Met Ala Leu Glu Lys Gln Arg Thr Ala Gly Lys Lys Ala Glu 115 120 125 ctt att gcc gtt gca gat gac gta tca gta ggt cgt aag aag agc ggt 432 Leu Ile Ala Val Ala Asp Asp Val Ser Val Gly Arg Lys Lys Ser Gly 130 135 140 aag gtc gga cgt cgt ggt ttg tct ggt act gtt ctt gtt cac aaa atc 480 Lys Val Gly Arg Arg Gly Leu Ser Gly Thr Val Leu Val His Lys Ile 145 150 155 160 gct ggt gca gct gcc gcc aga ggg tta cct ttg gaa gcc gtt acg acc 528 Ala Gly Ala Ala Ala Ala Arg Gly Leu Pro Leu Glu Ala Val Thr Thr 165 170 175 att gct aag gct gct att gac aat ttg gtt agt atc ggt gct tca ctc 576 Ile Ala Lys Ala Ala Ile Asp Asn Leu Val Ser Ile Gly Ala Ser Leu 180 185 190 gct cac gtt cac gtc cct ggt cat gag cca att gca aaa gaa gat gaa 624 Ala His Val His Val Pro Gly His Glu Pro Ile Ala Lys Glu Asp Glu 195 200 205 atg aaa cat gat gaa atg gaa ctt gga atg ggt att cac aat gaa cct 672 Met Lys His Asp Glu Met Glu Leu Gly Met Gly Ile His Asn Glu Pro 210 215 220 gga tgc aag cgt att tcc cct att ccc tct att gat gac cta att gct 720 Gly Cys Lys Arg Ile Ser Pro Ile Pro Ser Ile Asp Asp Leu Ile Ala 225 230 235 240 cag atg ctt aag caa atg ttg gat caa tcc gac aag gac cgt gcc tat 768 Gln Met Leu Lys Gln Met Leu Asp Gln Ser Asp Lys Asp Arg Ala Tyr 245 250 255 gta aag att gag ggt gac gat gaa gta gtc tta ctt atg aat aac ctt 816 Val Lys Ile Glu Gly Asp Asp Glu Val Val Leu Leu Met Asn Asn Leu 260 265 270 ggt ggt ctt tcc atg ctt gaa ttc agt gcc att agc cac aag gtg aag 864 Gly Gly Leu Ser Met Leu Glu Phe Ser Ala Ile Ser His Lys Val Lys 275 280 285 gaa gca ttg gct aaa gaa tac aaa atc aac ccc gtt cgc atc ttt gcc 912 Glu Ala Leu Ala Lys Glu Tyr Lys Ile Asn Pro Val Arg Ile Phe Ala 290 295 300 ggt cca ttt acc acc agt ttg aat ggc ttg ggt ttc ggt atc act ttg 960 Gly Pro Phe Thr Thr Ser Leu Asn Gly Leu Gly Phe Gly Ile Thr Leu 305 310 315 320 ctc cgt acc act gac cgc gtc aaa gtc gag ggc gaa gaa tac tct ttg 1008 Leu Arg Thr Thr Asp Arg Val Lys Val Glu Gly Glu Glu Tyr Ser Leu 325 330 335 gtt gat ttg att gac caa cct gtt gaa gct atc gga tgg cct ttg tgt 1056 Val Asp Leu Ile Asp Gln Pro Val Glu Ala Ile Gly Trp Pro Leu Cys 340 345 350 caa ccc tct gac ttg aag tcc aaa aac aag att ggc aat gtc agc atc 1104 Gln Pro Ser Asp Leu Lys Ser Lys Asn Lys Ile Gly Asn Val Ser Ile 355 360 365 gag gag ggt cag aag gat gtc aag tct ccc gtt act gtc gat aag gag 1152 Glu Glu Gly Gln Lys Asp Val Lys Ser Pro Val Thr Val Asp Lys Glu 370 375 380 aag gtt cgt cag gcg att gtc aat tcg atg gag aat ctc atc aaa gca 1200 Lys Val Arg Gln Ala Ile Val Asn Ser Met Glu Asn Leu Ile Lys Ala 385 390 395 400 gag cct aaa att aca aag ttc gat acg atg gct ggt gat ggt gac tgt 1248 Glu Pro Lys Ile Thr Lys Phe Asp Thr Met Ala Gly Asp Gly Asp Cys 405 410 415 ggt act act ttg aag cgt ggt gct gaa ggt gtt ttg aag ttt gtt aaa 1296 Gly Thr Thr Leu Lys Arg Gly Ala Glu Gly Val Leu Lys Phe Val Lys 420 425 430 tcc gac aaa ttc tct gac gat cct att cgt att gtt cgt gat atc gca 1344 Ser Asp Lys Phe Ser Asp Asp Pro Ile Arg Ile Val Arg Asp Ile Ala 435 440 445 gat gtt att gaa gac aat atg gat ggt aca tct ggt gct ttg tac gcc 1392 Asp Val Ile Glu Asp Asn Met Asp Gly Thr Ser Gly Ala Leu Tyr Ala 450 455 460 att ttc ttc cat ggc ttt gcg aag ggc atg aaa gac acc ttg gag aag 1440 Ile Phe Phe His Gly Phe Ala Lys Gly Met Lys Asp Thr Leu Glu Lys 465 470 475 480 agc aag gac att tca tct aag aca tgg gct gct ggt ttg aag gtt gct 1488 Ser Lys Asp Ile Ser Ser Lys Thr Trp Ala Ala Gly Leu Lys Val Ala 485 490 495 ctt gat act ctt ttc aag tat act ccc gct cgt cct ggt gac agc act 1536 Leu Asp Thr Leu Phe Lys Tyr Thr Pro Ala Arg Pro Gly Asp Ser Thr 500 505 510 atg tgt gat gct ctt gtt cca ttt gtc gaa aca ttt gtt aaa act aat 1584 Met Cys Asp Ala Leu Val Pro Phe Val Glu Thr Phe Val Lys Thr Asn 515 520 525 gat ctt aat gct gcc gta gag gag gct cgt aaa ggt gct gat gct act 1632 Asp Leu Asn Ala Ala Val Glu Glu Ala Arg Lys Gly Ala Asp Ala Thr 530 535 540 gca gat atg caa gcc aaa ctt gga cgt gct gtc tac gtt ggt gat gat 1680 Ala Asp Met Gln Ala Lys Leu Gly Arg Ala Val Tyr Val Gly Asp Asp 545 550 555 560 gtt aaa gtt ccc gat gcc ggc gct ctt ggt gtc gtt gca att gtc gaa 1728 Val Lys Val Pro Asp Ala Gly Ala Leu Gly Val Val Ala Ile Val Glu 565 570 575 gga ttt acg aaa taa 1743 Gly Phe Thr Lys 580 2 580 PRT Schizosaccharomyces pombe 2 Met Asp Lys His Phe Ile Asn Asp Pro Glu Val Leu Val Leu Asp Gly 1 5 10 15 Leu Lys Ser Leu Ala Asp Met Asn Lys Thr Leu Thr Val His Glu Glu 20 25 30 Gly Lys Phe Ile Tyr Phe His Asp Tyr Asn Lys Lys Asn Val Ser Val 35 40 45 Ile Ser Gly Gly Gly Ala Gly His Glu Pro Thr His Ser Ser Phe Val 50 55 60 Gly Lys Gly Met Leu Thr Ala Ala Val Ser Gly Ser Ile Phe Ala Ser 65 70 75 80 Pro Ser Ser Lys Gln Ile Tyr Thr Gly Ile Lys Gln Val Glu Ser Glu 85 90 95 Ala Gly Thr Leu Val Ile Cys Lys Asn Tyr Thr Gly Asp Ile Leu His 100 105 110 Phe Gly Met Ala Leu Glu Lys Gln Arg Thr Ala Gly Lys Lys Ala Glu 115 120 125 Leu Ile Ala Val Ala Asp Asp Val Ser Val Gly Arg Lys Lys Ser Gly 130 135 140 Lys Val Gly Arg Arg Gly Leu Ser Gly Thr Val Leu Val His Lys Ile 145 150 155 160 Ala Gly Ala Ala Ala Ala Arg Gly Leu Pro Leu Glu Ala Val Thr Thr 165 170 175 Ile Ala Lys Ala Ala Ile Asp Asn Leu Val Ser Ile Gly Ala Ser Leu 180 185 190 Ala His Val His Val Pro Gly His Glu Pro Ile Ala Lys Glu Asp Glu 195 200 205 Met Lys His Asp Glu Met Glu Leu Gly Met Gly Ile His Asn Glu Pro 210 215 220 Gly Cys Lys Arg Ile Ser Pro Ile Pro Ser Ile Asp Asp Leu Ile Ala 225 230 235 240 Gln Met Leu Lys Gln Met Leu Asp Gln Ser Asp Lys Asp Arg Ala Tyr 245 250 255 Val Lys Ile Glu Gly Asp Asp Glu Val Val Leu Leu Met Asn Asn Leu 260 265 270 Gly Gly Leu Ser Met Leu Glu Phe Ser Ala Ile Ser His Lys Val Lys 275 280 285 Glu Ala Leu Ala Lys Glu Tyr Lys Ile Asn Pro Val Arg Ile Phe Ala 290 295 300 Gly Pro Phe Thr Thr Ser Leu Asn Gly Leu Gly Phe Gly Ile Thr Leu 305 310 315 320 Leu Arg Thr Thr Asp Arg Val Lys Val Glu Gly Glu Glu Tyr Ser Leu 325 330 335 Val Asp Leu Ile Asp Gln Pro Val Glu Ala Ile Gly Trp Pro Leu Cys 340 345 350 Gln Pro Ser Asp Leu Lys Ser Lys Asn Lys Ile Gly Asn Val Ser Ile 355 360 365 Glu Glu Gly Gln Lys Asp Val Lys Ser Pro Val Thr Val Asp Lys Glu 370 375 380 Lys Val Arg Gln Ala Ile Val Asn Ser Met Glu Asn Leu Ile Lys Ala 385 390 395 400 Glu Pro Lys Ile Thr Lys Phe Asp Thr Met Ala Gly Asp Gly Asp Cys 405 410 415 Gly Thr Thr Leu Lys Arg Gly Ala Glu Gly Val Leu Lys Phe Val Lys 420 425 430 Ser Asp Lys Phe Ser Asp Asp Pro Ile Arg Ile Val Arg Asp Ile Ala 435 440 445 Asp Val Ile Glu Asp Asn Met Asp Gly Thr Ser Gly Ala Leu Tyr Ala 450 455 460 Ile Phe Phe His Gly Phe Ala Lys Gly Met Lys Asp Thr Leu Glu Lys 465 470 475 480 Ser Lys Asp Ile Ser Ser Lys Thr Trp Ala Ala Gly Leu Lys Val Ala 485 490 495 Leu Asp Thr Leu Phe Lys Tyr Thr Pro Ala Arg Pro Gly Asp Ser Thr 500 505 510 Met Cys Asp Ala Leu Val Pro Phe Val Glu Thr Phe Val Lys Thr Asn 515 520 525 Asp Leu Asn Ala Ala Val Glu Glu Ala Arg Lys Gly Ala Asp Ala Thr 530 535 540 Ala Asp Met Gln Ala Lys Leu Gly Arg Ala Val Tyr Val Gly Asp Asp 545 550 555 560 Val Lys Val Pro Asp Ala Gly Ala Leu Gly Val Val Ala Ile Val Glu 565 570 575 Gly Phe Thr Lys 580 3 33 DNA Artificial Sequence Synthetically generated primer 3 gccggtacca ggagctaaaa tatggataag cac 33 4 33 DNA Artificial Sequence Synthetically generated primer 4 gccaagctta aaatttcgta tccaattatt tcg 33 

1. A method for producing dihydroxyacetone-3-phosphate by a catalytic reaction of dihydroxyacetone with the bacterial cells transformed with the gene encoding a dihydroxyacetone kinase or the dihydroxyacetone kinase produced by said bacterium.
 2. A method according to claim 1, wherein said dihydroxyacetone kinase is derived from yeast Schizosaccharomyces.
 3. A method according to claim 1, wherein said dihydroxyacetone kinase is derived from Schizosaccharomyces pombe.
 4. A method according to claim 1, wherein said bacterium is Escherichia coli.
 5. A method according to claims 1 to 4, wherein said enzymatic reaction is performed under the conditions of a regeneration system for ATP. 